Coronary artery disease (CAD) is the leading cause of morbidity and mortality in most developed countries. Numerous markers and tests for identifying individuals at risk are available, among them blood tests for lipid markers such as total cholesterol and cholesterol bound to various circulating proteins. Based on the outcome of such testing, appropriate prophylactic or therapeutic measures including dietary modification and exercise can be initiated to forestall or reverse progression to more severe CAD.
Plasma lipoproteins are carriers of lipids from the sites of synthesis and absorption to the sites of storage and/or utilization. Lipoproteins are spherical particles with triglycerides and cholesterol esters in their core and a layer of phospholipids, nonesterified cholesterol and apolipoproteins on the surface. They are categorized into five major classes based on their hydrated density as very large, triglyceride-rich particles known as chylomicrons (less than 0.95 g/ml), very low density lipoproteins (VLDL, 0.95 to 1.006 g/ml), intermediate-density lipoproteins (IDL, 1.006 to 1.019 g/ml), low-density lipoproteins (LDL, 1.019 to 1.063 g/ml) and, high-density lipoproteins (HDL, 1.063 to 1.210 g/ml). (Osborne and Brewer, Adv. Prot. Chem. 31:253-337 (1977); Smith, L. C. et al. Ann Rev. Biochem., 47:751-777 (1978)).
Apolipoproteins are protein components of lipoproteins with three major functions: (1) maintaining the stability of lipoprotein particles, (2) acting as cofactors for enzymes that act on lipoproteins, and (3) removing lipoproteins from circulation by receptor-mediated mechanisms. The four groups of apolipoproteins are apolipoproteins A (Apo A), B (Apo B), C (Apo C) and E (Apo E). Each of the three groups A, B and C consists of two or more distinct proteins. These are for Apo A: Apo A-I, Apo A-II, and Apo A-IV, for Apo B: Apo B-100 and Apo B-48; and for Apo C: Apo C-I, Apo C-II and Apo C-III. Apo E includes several isoforms. Each class of lipoproteins includes a variety of apolipoproteins in differing proportions with the exception of LDL, which contains Apo B-100 as the sole apolipoprotein. Apo A-I and Apo A-II constitute approximately 90 percent of the protein moiety of HDL whereas Apo C and Apo E are present in various proportions in chylomicrons, VLDL, IDL and HDL. Apo B-100 is present in LDL, VLDL and IDL. Apo B-48 resides only in chylornicrons and so called chylomicron remnants (Kane, J. P., Method. Enzymol. 129:123-129 (1986)).
Total plasma or serum cholesterol (C) has traditionally been the primary screening and indicator of CAD, but the emphasis has recently shifted to serum lipoprotein profiles including HDL, LDL, VLDL, lipoprotein A and particularly to the LDL/HDL or Total C/HDL ratios which have shown better correlations with incidence and severity of CAD. In contrast to the atherogenic potential of LDL, VLDL and VLDL remnants, HDL are inversely correlated with CHD, so that individuals with low concentrations of HDL-C have an increased incidence of CHD (Gordon, T. et al., Am. J. Med., 62:707-714 (1977); Miller, N. E. et al., Lancet, 1:965-968 (1977); Miller, G. J. and Miller, N. E., Lancet, 1:16-19 (1975)).
A large number of manual and automated methods are available for screening and monitoring of these markers. All of these tests, however, require either venous blood drawn by syringe or, in some cases, capillary blood obtained by needle prick. Both methods are invasive and unpleasant to many individuals and are best performed by trained professional personnel, preferably in doctor""s office, to minimize erroneous results. Handling and disposal of blood products also involves potential hazards from infectious agents and pathogens.
It is thus highly desirable to provide safer alternative specimens not requiring invasive procedures. Furthermore, the ideal analytical method or device should provide rapid and reliable results for point of collection (xe2x80x9cPOCxe2x80x9d) diagnosis at low cost.
Most analytes that appear in serum also appear in saliva, but at levels that are a fraction of their level in serum. The transport of an analyte into saliva can be by intracellular (diffusion or passive transport) or extracellular (active transport) transport. Materials that are lipid soluble enter saliva by diffusion through cellular compartments. Haekcel, Ann. N.Y. Acad. Sci. 694, 128-142 (1993).
Saliva has not been exploited as a diagnostic fluid because of the many problems associated with adapting it to assay form. For example, it is difficult to collect sufficient sample: Most tests require collection of at least 1 ml of saliva because there is considerable loss during filtration and handling. This requires an average of 3-5 minutes of salivation, which most people are not willing to do. The average flow rate for 95% of young men is 0.35-0.38 ml/min. (K. Diem, et al (ed) Scientific Tables (Ciba-Geigy Pharmaceuticals 1970) p. 643. Moreover, the handling of saliva samples to prepare them for assay is both tedious and unpleasant. Saliva generally has to be filtered to remove the mucopolysaccharides and allow flow and handling. Available collection devices utilize cotton pads to absorb saliva in the mouth. The pad thus acts to collect and process the saliva, preparing it for assay. The pad is then placed in a volume of fluid containing preservatives and shipped to the laboratory for analysis. The preservative fluid prevents quantitation by making it impossible to know how much saliva, if any, was collected and added to the preservative. When the device reaches the lab the technicians must remove the pad and mucopolysaccharides either by centrifugation or filtration. This is a time consuming and unpleasant job. The small amount of saliva sample and low level of analyte in saliva usually means that the saliva sample cannot be analyzed by an autoanalyzer, but must be assayed in a high sensitivity Elisa or RIA, both of which are labor-intensive tests.
Many studies of saliva have shown that the levels of analytes vary with the secreting gland and the method of collection (e.g. stimulated flow versus normal flow). For reviews see Saliva as a Diagnostic Fluid (D. Malmud and L. Tobak, Eds., Ann. N.Y. Acad. Sci. Vol. 694 D (1993) and J. O. Tenuvuo (ed) Human Saliva: Clinical Chemistry and Microbiology (CRC Press Inc. 1989) vol. I and II). Thus, one presumes that the significant variations in lipid levels reported in saliva are in large part due to collection method. Levels of cholesterol are also low, with cholesterol levels of about 1/400 and about 1/50 of that seen in serum. Bronislaw, et al., xe2x80x9cLipids of Saliva and Salivary Concretions,xe2x80x9d in Human Saliva: Clinical Chemistry and Microbiology (CRC Press Inc. 1989) vol. II, 121-145). Thus the level in an individual sample is too low for conventional serum assays in the routine assay of lipids in saliva, therefore either requiring the use of sensitive immunoassays or a larger quantity of saliva. J. C Touchstone, et al., xe2x80x9cQuantitation of Cholesterol in Biological.xe2x80x9d in Adv. Thin Layer Chromatogr., Proc. Bienn. Symp. Meeting Date 1980, (Wiley and Sons 2nd ed. 1982) measured total cholesterol and lipids. Moreover, there is a variation in levels depending on the time of day and from day to day (less than 8%), with levels highest in morning specimens and lower throughout the day, suggesting that saliva testing of cholesterol be done at the same time of the day.
Another problem with using saliva is that saliva is heavily contaminated with the oral flora. Available collection devices provide high levels of preservatives to retard growth of bacteria but, unless the sample is carefully preserved (e.g. by freezing), samples often become putrefied and laboratory technicians avoid processing saliva. Furthermore, high levels of preservatives can interfere in many assays. Saliva also contains many proteins and enzymes of both salivary and bacterial origins. Over time these enzymes and proteins can interact with the analytes of interest and make the assay of some analytes impossible. Thus, as a rule, stored samples cannot be expected to yield accurate results unless the storage additives and conditions are optimized for the analyte.
The literature reports that, while cholesterol is present in saliva, the levels vary greatly. For example, 5.6 mg/L average was reported by B. Larsson, et al, xe2x80x9cLipids in Human Salivaxe2x80x9d in Archs. Oral. Biol. 41(1), 105-110 (1996); 15 mg/L average was reported for both the parotid and submandibular glands saliva output by Slomiany, et al, J. Dent. Res. 61(1), 24-27 (1983); and 69 mg/L was reported by Rabinowitz, et al, Arch. Oral. Biol. 20(7), 403-406 (1975).
As noted above, the ratio of LDL:HDL ratio is an established predictor of the risk of coronary artery disease. The recent NCEP guidelines call for use of ratio rather than total cholesterol. It has been reported that men with acceptable total cholesterol levels but ratios of LDL:HDL above 3.5 were 50% more likely to have coronary heart disease than their counterparts with lower ratios. It is a matter of time before total cholesterol is supplanted by ratios.
Immunoassays for lipoproteins associated with HDL and LDL have been shown to correlate with the measurement of cholesterol ratios in these two fractions. N. Rifai, et al (ed) Laboratory Measurement of Lipids, Lipoproteins and Apolipoproteins (AACC Press 1994) p. 114. The results correlate with the methods where HDL and LDL fractions are physically separated and measured (Laboratory Measurement of Lipids, Lipoproteins and Apolipoproteins. 1994. N. Rifai and R. Warnick Eds. AACC Press.)
It is not known if the proteins with which salivary cholesterol is associated are the same as those in serum, i.e. ApoAI and ApoBII. It is clear from all studies (Belmont) (Mandel, et al, Arch. Oral. Biol. 14(2), 231-233 (1969)) that salivary lipids are secreted by the glands in conjunction with lipoproteins(s). Slomiany et al also demonstrated that the lipids in saliva are associated with proteins. There is no published literature, however, on the origin of the lipids or their physical state in salivaxe2x80x9d (Larsson et al). Thus, from the early literature, it is not clear whether the salivary lipids are synthesized de novo in the salivary glands or are derived from serum; and, if they are serum derived, if the salivary apolipoproteins are the same as the apolipoproteins associated with LDL and HDL in serum. There are other salivary glycoproteins also associated with the lipid. It is not clear from the literature whether the structure of the lipid particles in saliva is the same as those in serum and whether the conformation of the apolipoproteins is the same in saliva as in serum. Rabinowitz suggests that lipids secreted by the glands are secreted associated with lipoprotein. He demonstrated that the lipid levels drop in stimulated saliva but retain the same ratio to one another. Larsson reports that salivary lipoprotein fractions are of much higher density than serum lipoproteins and concludes that the salivary lipids are differently aggregated.
Various studies have indicated that the saliva levels of cholesterol show a gross correlation with serum cholesterol levels (Lochner, A. Dissertation Abstract International (1985) Vol. 46, #5B). It has also been observed that there is a positive correlation between persons with hypercholesteremia. Slorniany, et al, Arch. Oral. Biol. 27(10), 803-808 (1982) and Murty, et al, RCS Med. Sci. 10(5), 359 (1982). The paucity of studies correlating serum and saliva cholesterol may be due to the fact that the available methods for assaying cholesterol and thus correlating serum and saliva have been too insensitive. The enzymic and chromatographic methods of detecting cholesterol rely on high levels not available in saliva. Thus, these methods require large amounts of saliva and studies on lipids have generally been done on pools. Measurement of cholesterol in saliva is further complicated because saliva contains high amounts of peroxidase, an enzyme component of some cholesterol assays.
It is therefore an object of the present invention to provide a non-invasive, non-instrumental, accurate, simple and cost-effective means for determination of a marker for CAD, HDL, LDL, and/or the ratio of LDL:HDL.
A method and kit has been developed to detect the levels of apolipoproteins A-1 and B in saliva, which is correlated with the levels of HDL and LDL in serum, respectively. In unstimulated saliva, the ratio of Apo A to Apo B is correlated with the ratio of HDL to LDL in serum. In stimulated saliva the levels of Apo B normalized to albumin correlate with both serum Apo B and serum LDL. The high degree of correlation in combination with a simple, quick test that can be performed at the site of collection provides a cost effective, patient friendly means to monitor an individual""s risk of heart disease. In the preferred embodiment, saliva production is stimulated by means such as breath mint or tart solution (such as lemon) and the effect of dilution controlled by reference to albumin. In the most preferred embodiment, the assay is an immunoassay performed using the Serex laminated strip format as described in U.S. Pat. Nos. 5,710,009, 5,500,375, and 5,451,504. These strips are advantageous since they serve as the collection and assay device, greatly simplifying handling, as the sample is applied directly to the strip test and processed as an integral part of the analytical procedure. This method requires less than 200 microliters, which should be available in the average person""s mouth at any time. Additional saliva production can be obtained, however, using breath mints or a tart juice such as lemon juice. The assay of saliva at POC will eliminates the need for preservatives to store the sample and entirely avoids the problem with contamination by oral flora, since the assay can be completed within 10 minutes of saliva collection.